Organelle Transport in Cultured &lt;em&gt;Drosophila&lt;/em&gt; Cells: S2 Cell Line and Primary Neurons.

Lü, Wen; Castillo, Urko del; Gelfand, Vladimir I.

doi:10.3791/50838

Cited by 20 publications

(40 citation statements)

References 28 publications

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“…In our search for proteins that mediate dynein-dependent sorting of microtubules in cell processes, we first conducted a candidate-based RNA interference (RNAi) screen in Drosophila S2 cells. S2 cells treated with 2.5 μM of Cytochalasin D (CytoD), an F-actin severing drug, form long microtubule-filled processes (17). Importantly, processes in CytoD treated cells contained abundant cortical actin (Fig.…”

Section: Resultsmentioning

confidence: 99%

Kinetochore protein Spindly controls microtubule polarity inDrosophilaaxons

Castillo¹,

Müller

Gelfand³

2020

Preprint

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24Microtubule polarity in axons and dendrites defines the direction of intracellular transport 25 in neurons. Axons contain arrays of uniformly polarized microtubules with plus-ends 26 facing the tips of the processes (plus-end-out), while dendrites contain microtubules with 27 minus-end-out orientation. It has been shown that cytoplasmic dynein, targeted to cortical 28 actin, removes minus-end-out microtubules from axons. Here we have identified Spindly, 29 a protein known for recruitment of dynein to kinetochores in mitosis, as a key factor 30 required for dynein-dependent microtubule sorting in axons of Drosophila neurons. 31Depletion of Spindly affects polarity of axonal microtubules in vivo and in primary neuronal 32cultures. In addition to these defects, depletion of Spindly in neurons causes major 33 collapse of axonal patterning in the third-instar larval brain as well as severe coordination 34 impairment in adult flies. These defects can be fully rescued by full-length Spindly, but 35 not by variants with mutations in its dynein-binding site. Biochemical analysis 36 demonstrated that Spindly binds F-actin, suggesting that Spindly serves as a link between 37 dynein and cortical actin in axons. Therefore, Spindly plays a critical role during 38 neurodevelopment by mediating dynein-driven sorting of axonal microtubules. 39 40 Significance Statement 41Neurons send and receive electrical signals through long microtubule-filled neurites called 42 axons and dendrites. One of the main structural differences between axons and dendrites 43 is how their microtubules are organized. Axons contains microtubules with their plus-ends 44 out while microtubules in dendrites are organized with mixed or plus-end-in orientation. 45Dynein, the main minus-end microtubule motor, anchored to cortical actin filaments in the 46 axons is responsible for the uniform microtubule polarity in axons. However, it is unknown 47 how dynein is recruited to the actin cortex in axons. The major finding of this work is that 48Spindly, a protein involved in anchoring dynein to kinetochores during cell division, has a 49 second important function in interphase cells recruiting dynein to the actin cortex in axons. 50 51 Introduction 52Neurons are post-mitotic cells that transmit electrical signals through long neurites called 53 axons and dendrites. Electric signals captured by dendrites are sent unidirectionally 54 through axons and transmitted to other cells. The polarity of microtubules is strikingly 55 different in these two types of neuronal processes. Axons contain microtubules oriented 56 predominantly with their plus-ends-out, while dendrites contain a large fraction of minus-57 end-out microtubules (1, 2). Failure in establishing correct polarity of microtubules results 58 in defects of cargo sorting (3). The differences in microtubule orientation between axons 59 and dendrites are gradually established during development. In cultured early stage 60 neurons, non-polarized neurites contain microtubules with mixed orientation. Later the 61...

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Section: Resultsmentioning

confidence: 99%

Kinetochore protein Spindly controls microtubule polarity inDrosophilaaxons

Castillo¹,

Müller

Gelfand³

2020

Preprint

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“…lens at two different focal planes that are 5-10 μm apart (at the cortex and underneath the cortex, respectively). Images were analyzed in Fiji and DiaTrack 3.04 Pro (73,74), with a maximum particle jump distance of 3.75 μm/s. Velocities were obtained using Analysis > Distribution of Speed (Track-Based) in DiaTrack 3.04 Pro.…”

Section: Methodsmentioning

confidence: 99%

“…Young mated female adults were fattened for ∼48-72 h and dissected in 1× dissecting saline [9.9 mM Hepes (pH 7.5), 137 mM NaCl, 5.4 mM KCl, 0.17 mM NaH 2 PO 4 , 0.22 mM KH 2 PO 4 , 3.3 mM glucose, 43.8 mM sucrose]. Ovaries were placed in lysis buffer on ice [50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 2 mM MgCl 2 , 1 mM PMSF, 1× CLP (10 μg/mL chymostatin, leupeptin, pepstatin A), 0.5% Triton X-100] and mechanically homogenized using pellet pestles in matching microtubes (74). Ovary homogenates were spun for 10 min at >20,000 × g at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

Lü

Winding

Lakonishok

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Cytoplasmic streaming in Drosophila oocytes is a microtubulebased bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule-microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants.kinesin-1 | microtubules | cytoplasmic streaming | Drosophila | axis determination

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“…Microtubule sliding is only active in young growing neurons, and becomes undetectable as they mature [15, 16]. Downregulation of microtubule sliding is not caused by inhibition of kinesin-1, since the kinesin-1-dependent organelle transport is still active in mature neurons [9, 15, 16, 36]. We demonstrated that a “mitotic” kinesin Pavarotti (a kinesin-6 family member known in other organisms as MKLP1, CHO1 or KIF20A) is a potent negative regulator of kinesin-1-driven sliding of cytoplasmic microtubules [17], most likely crosslinking anti-parallel microtubules in neurons.…”

Section: Developmental Regulation Of Microtubule Slidingmentioning

confidence: 99%

Moonlighting Motors: Kinesin, Dynein, and Cell Polarity

Lü

Gelfand

2017

Trends in Cell Biology

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In addition to their well-known role in transporting cargoes in the cytoplasm, microtubule motors organize their own tracks, microtubules. While this function is mostly studied in the context of cell division, it is essential for microtubule organization and generation of cell polarity in interphase cells. Kinesin-1, the most abundant microtubule motor, plays a role in the initial formation of neurites. This review describes the mechanism of kinesin-1-driven microtubule sliding and discusses its biological significance in neurons. Recent studies describing the interplay between kinesin-1 and cytoplasmic dynein in translocation of microtubules are discussed. In addition, we evaluate recent work exploring the developmental regulation of microtubule sliding during axonal outgrowth and regeneration. Collectively, the discussed works suggest that sliding of interphase microtubules by motors is a novel force-generating mechanism that reorganizes the cytoskeleton and drives shape change and polarization.

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Organelle Transport in Cultured <em>Drosophila</em> Cells: S2 Cell Line and Primary Neurons.

Cited by 20 publications

References 28 publications

Kinetochore protein Spindly controls microtubule polarity inDrosophilaaxons

Kinetochore protein Spindly controls microtubule polarity inDrosophilaaxons

Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

Moonlighting Motors: Kinesin, Dynein, and Cell Polarity

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